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Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay
用于超氧化物歧化酶(SOD)活性试验的拟南芥细胞提取物的制备    

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Abstract

Superoxide dismutase (SOD) acts as a primary defence against reactive oxygen species (ROS) by converting O2- to O2 and H2O2. Members of this enzyme family include CuZnSOD, MnSOD and FeSOD. Most eukaryotes harbor CuZnSOD and MnSOD, and FeSOD is found in plants and prokaryotes. This protocol is to demonstrate how to prepare the cellular extract for the identification and characterization of SODs in planta.

Keywords: SOD(草地), Activity Assay(活性测定), N(n)

Materials and Reagents

  1. Nitroblue tetrazolium (NBT) (Sigma-Aldrich, catalog number: N6876 )
  2. Riboflavin (Sigma-Aldrich, catalog number: R4500 )
  3. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  4. KCN (Sigma-Aldrich, catalog number: 60178 )
  5. H2O2 (Sigma-Aldrich, catalog number: 349887 )
  6. NBT solution (see Recipes)
  7. Grinding buffer (see Recipes)
  8. Riboflavin solution (see Recipes)
  9. KCN solution (see Recipes)
  10. H2O2 solution (see Recipes)

Equipment

  1. A light box (white light)
  2. Centrifuge (Heraecus, Biofuge fresco)
  3. Protein gel cassette

Procedure

  1. Arabidopsis cellular extract preparation
    1. Arabidopsis seedlings were grown at 23 °C with 16 h of light at 60–100 μmol m-2 s-1. Nine-day-old seedlings were collected and weighted.
    2. Seedlings were homogenized with ice-cold Grinding buffer (tissue weight/buffer volume = 1 mg/3 μl).
      Note that the tissue and extract should be kept at 4 °C during all extraction processes.
    3. Centrifuge at 16,000 x g at 4 °C for 10 min.
    4. The supernatant is the resulting cellular extract, and the amount of protein was quantified by Bradford method (1976).
  2. SOD activity staining
    1. Proteins or cellular extract (15 to 25 μg) was subjected to 10% native-PAGE at 4 °C.
    2. Wash the gel with distilled water for 3 times.
    3. Incubate with NBT solution in dark with shaking for 15 min at room temperature (RT).
    4. Pour off the NBT solution, wash the gel with distilled water for 3 times.
    5. Incubate with Riboflavin solution in dark with shaking for 15 min at RT.
    6. Pour off the Riboflavin solution, wash the gel with distilled water for 3 times.
    7. Gel was illuminated with a white-light box for 10-15 min at RT. During illumination, immerse gel in a thin layer of distilled water to avoid drying the gel.
    8. Under light exposure, the riboflavin is reduced then leading the production of O2-. NBT is reduced by O2- to form formazan, a dark blue color precipitate. The enriched SOD activity scavenges the O2- to prevent the formation of formazan, thus, the white SOD activity bands appear in the blue background.
  3. Identification of different SOD species (Figure 1)
    1. KCN treatment: KCN inhibits the CuZnSOD activity only.
      All procedures are the same as SOD activity staining processes except the addition of KCN to final 8 mM in Riboflavin solution.
    2. H2O2 treatment: H2O2 inhibits both CuZnSOD and FeSOD activities.
      After native-PAGE and prior to NBT staining of SOD activity staining processes, soak the gel with 8 mM H2O2 solution for 30 min with shaking at room temperature. Wash the gel with distilled water for 3 times, and follow the remaining processes of SOD activity staining.


Figure 1. SOD activity verification in Arabidopsis thaliana. KCN is an inhibitor of CuZnSOD activity, whereas H2O2 inhibits both CuZnSOD and FeSOD activities. MnSOD activity is not inhibited by either treatment.

Recipes

  1. Grinding buffer
    150 mM Tris (pH 7.2)
  2. NBT solution
    0.1% NBT dissolved in distilled water.
    Store in 4 °C in dark
  3. Riboflavin solution (freshly prepare before use)
    28 μM riboflavin and 28 mM TEMED in 0.1 M potassium phosphate buffer (pH 7.0).
  4. 2 N KCN solution
    KCN in distilled water. Store in 4 °C.
  5. 8 mM H2O2 solution (freshly prepare before use)
    Add 27 μl H2O2 (35%) into 30 ml 0.1 M potassium phosphate buffer (pH 7.0).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Kuo, W. Y., Huang, C. H., Liu, A. C., Cheng, C. P., Li, S. H., Chang, W. C., Weiss, C., Azem, A. and Jinn, T. L. (2013). CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts. New Phytol 197(1): 99-110.

简介

超氧化物歧化酶(SOD)通过将O 2转化为O 2和H 2 O而作为对活性氧(ROS)的主要防御 2 。 该酶家族的成员包括CuZnSOD,MnSOD和FeSOD。 大多数真核生物具有CuZnSOD和MnSOD,FeSOD存在于植物和原核生物中。 该协议是为了说明如何准备细胞提取物用于在植物中鉴定和表征SOD。

关键字:草地, 活性测定, n

材料和试剂

  1. 硝基蓝四唑(NBT)(Sigma-Aldrich,目录号:N6876)
  2. 核黄素(Sigma-Aldrich,目录号:R4500)
  3. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  4. KCN(Sigma-Aldrich,目录号:60178)
  5. (Sigma-Aldrich,目录号:349887)。< br />
  6. NBT解决方案(参见配方)
  7. 研磨缓冲器(参见配方)
  8. 核黄素溶液(见配方)
  9. KCN解决方案(参见配方)

  10. 解决方案(参见配方)。

设备

  1. 灯箱(白光)
  2. 离心机(Heraecus,Biofuge fresco)
  3. 蛋白凝胶盒

程序

  1. 拟南芥细胞提取物制备
    1. 拟南芥幼苗在23℃,16小时光照下生长 60-100μmolm -2 -S sup -1 -1 。 收集九天的幼苗并称重
    2. 幼苗用冰冷的研磨缓冲液(组织重量/缓冲液体积= 1mg /3μl)匀浆。
      请注意,在所有提取过程中,组织和提取物应保持在4°C 。
    3. 在4℃下以16,000×g离心10分钟
    4. 上清液是所得的细胞提取物,并且通过Bradford方法(1976)定量蛋白质的量。
  2. SOD活性染色
    1. 将蛋白质或细胞提取物(15至25μg)在4℃下进行10%天然PAGE
    2. 用蒸馏水洗涤凝胶3次。
    3. 在黑暗中与NBT溶液孵育,在室温(RT)下摇动15分钟
    4. 倒出NBT溶液,用蒸馏水洗涤凝胶3次。
    5. 与核黄素溶液在黑暗中孵育15分钟,在室温摇动
    6. 倒出核黄素溶液,用蒸馏水洗涤凝胶3次。
    7. 凝胶 在室温下用白光盒照射10-15分钟。 在照明期间,将凝胶浸入薄层 蒸馏水以避免干燥凝胶
    8. 在光下 暴露,核黄素减少,然后导致O 2 - 的产生。 NBT还原O 2 - 形成甲an,深蓝色沉淀。 富集的SOD活性清除O 2 - 以防止其形成 甲an,因此,白色SOD活性带出现在蓝色 背景
  3. 不同SOD物种的鉴定(图1)
    1. KCN处理:KCN仅抑制CuZnSOD活性 所有程序与SOD活性染色过程相同,只是在核黄素溶液中加入KCN至最终8mM。
    2. H 2 O 2处理:H 2 O 2抑制CuZnSOD和FeSOD活性。
      在天然PAGE和在SOD活性染色过程的NBT染色之前,用8mM H 2 O 2 Sub 2溶液在室温下振摇将凝胶浸泡30分钟。 用蒸馏水洗涤凝胶3次,并遵循剩余的SOD活性染色过程。


图1.拟南芥中的SOD活性验证。 KCN是CuZnSOD活性的抑制剂,而H sub 2 O >抑制CuZnSOD和FeSOD活性。 任何一种处理都不会抑制MnSOD活性。

食谱

  1. 研磨缓冲器
    150mM Tris(pH7.2)
  2. NBT解决方案
    0.1%NBT溶于蒸馏水中。
    在4°C下储存在暗处
  3. 核黄素溶液(使用前新配制)
    28μM核黄素和28mM TEMED在0.1M磷酸钾缓冲液(pH 7.0)中
  4. 2 N KCN溶液
    KCN在蒸馏水中。 储存于4°C。
  5. 8mM H 2 O 2溶液(使用前新鲜制备)
    将27μlH 2 O 2 Sub(35%)加入到30ml 0.1M磷酸钾缓冲液(pH 7.0)中。

参考文献

  1. Bradford,M. M.(1976)。一种快速灵敏的微克定量方法 使用蛋白染料结合原理的蛋白质的量。 Anal Biochem 72:248-254。
  2. Kuo,W.,Huang,C. H.,Liu,A. C.,Cheng,C. P.,Li,S. H.,Chang,W. C.,Weiss,C.,Azem,A.and Jinn,T.L。 CHAPERONIN 20介导铁超氧化物歧化酶(FeSOD)活性,而不依赖于其共伴侣作用, 拟南芥叶绿体。 New Phytol 197(1):99-110。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kuo, W., Huang, C., Shih, C. and Jinn, T. (2013). Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay. Bio-protocol 3(13): e811. DOI: 10.21769/BioProtoc.811.
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gary jefferson
UBS
Is there any way to purchase SOD Gel. I read the article by Dr. Frank Shallenberger in "Second Opinion", January 2015, which described the significant results using SOD Gel. Unfortunately, I can't locate a source for purchasing it anywhere. Many thanks, Gary
1/7/2015 12:14:14 PM Reply
Tsung-Luo Jinn
Department of Life Science and Institute of Plant Biology, National Taiwan University

You only need is the SDS eliminated 12.5% PAGE (without SDS), which is good for in gel SOD activity assay, best,

1/7/2015 11:16:28 PM


Rashmi Mishra
M.S University
DOES SOD GETS DENATURED AT TEMPERATURES HIGHER THAN 4 DEGREE CELCIUS?
1/1/2015 1:16:57 AM Reply
Tsung-Luo Jinn
Department of Life Science and Institute of Plant Biology, National Taiwan University

Hi,
Please extract your sample with ice-cold buffer, and then run your gel in cold room,
Which can give you best result,
Sure, SOD may or may not denature when temp higher then 4C, different SOD from different sample do have it optimal reaction temp and pH value,
Best,

1/4/2015 12:17:37 AM